Petronia Carillo

The sucrose–trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P

Summary Trehalose-6-phosphate is a signal of sucrose status in plants and forms part of a homeostatic mechanism that maintains sucrose levels within a range that is appropriate for the cell type and stage of development.

Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

BackgroundReactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli.ResultsTo investigate this role further, we exposed wild type Arabidopsis thaliana plants and the double mutant npq1lut2 to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (1O2) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by 1O2 which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in npq1lut2 is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the flu mutant was the only biological system adopted to define the regulation of gene expression by 1O2. In this work, we propose the use of mutants accumulating 1O2 by mechanisms different from those activated in flu to better identify ROS signalling.ConclusionsWe propose that the lack of zeaxanthin and lutein leads to 1O2 accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to 1O2 accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed.

Nitrate reductase in durum wheat seedlings as affected by nitrate nutrition and salinity

The combined effects of nitrate (0, 0.1, 1, 10 mm) and salt (0, 100 mm NaCl) on nitrogen metabolism in durum wheat seedlings were investigated by analysis of nitrate reductase (NR) expression and activity, and metabolite content. High salinity (100 mm NaCl) reduced shoot growth more than root growth. The effect was independent of nitrate concentration. NR mRNA was present at a low level in both leaves and roots of plants grown in a nitrogen-free medium. NaCl increased NR mRNA at low nitrate, suggesting that chloride can mimic nitrate as a signal molecule to induce transcription in both roots and leaves. However, the level of NR protein remained low in salt-stressed plants, indicating an inhibitory effect of salt on translation of NR mRNA or an increase in protein degradation. The lower activity of nitrate reductase in leaves of high-nitrate treated plants under salinity suggested a restriction of NO3- transport to the shoot under salinity. Salt treatment promoted photorespiration, inhibiting carbohydrate accumulation in plants grown on low nitrate media. Under salinity free amino acids, in particular proline and asparagine, and glycine betaine could function as osmolytes to balance water potential within the cell, especially when nitrogen availability exceeded the need for growth.

Nitrogen metabolism in durum wheat under salinity: accumulation of proline and glycine betaine

We studied the effect of salinity on amino acid, proline and glycine betaine accumulation in leaves of different stages of development in durum wheat under high and low nitrogen supply. Our results suggest that protective compounds against salt stress are accumulated in all leaves. The major metabolites are glycine betaine, which preferentially accumulates in younger tissues, and proline, which is found predominantly in older tissues. Proline tended to accumulate early, at the onset of the stress, while glycine betaine accumulation was observed during prolonged stress. Nitrate reductase (NR) and glutamate synthase (GOGAT) are positively correlated with these compatible solutes: proline is associated with NR in the oldest leaves of high-nitrate plants and glycine betaine is associated with GOGAT in the youngest leaves of both low- and high-nitrate plants. In high-nitrate conditions proline accounts for more than 39% of the osmotic adjustment in the cytoplasmic compartments of old leaves. Its nitrogen-dependent accumulation may offer an important advantage in that it can be metabolised to allow reallocation of energy, carbon and nitrogen from the older leaves to the younger tissues. The contribution of glycine betaine is higher in young leaves and is independent of nitrogen nutrition.

Plant Genes for Abiotic Stress

Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50%. Plants as sessile organisms are constantly exposed to changes in environmental conditions. When these changes are rapid and extreme, plants generally perceive them as stresses. However stresses are not necessarily a problem for plants because they have evolved effective mechanisms to avoid or reduce the possible damages. The response to changes in environment can be rapid, depending on the type of stress and can involve either adaptation mechanisms, which allow them to survive the adverse conditions, or specific growth habitus to avoid stress conditions. In fact, plants can perceive abiotic stresses and elicit appropriate responses with altered metabolism, growth and development. The regulatory circuits include stress sensors, signalling pathways comprising a network of protein-protein interactions, transcription factors and promoters, and finally the output proteins or metabolites (table 1). A number of abiotic stresses such as extreme temperatures, high light intensity, osmotic stresses, heavy metals and a number of herbicides and toxins lead to over production of reactive oxygen species (ROS) including H2O2 causing extensive cellular damage and inhibition of photosynthesis. Normally, ROS are rapidly removed by antioxidative mechanisms, but this removal can be impaired by stresses themselves (Allan & Fluhr, 2007), causing a rise in their intracellular concentration and an increase of the damage. To prevent or repair these damages, plant cells use a complex defence system, involving a number of antioxidative stress-related defence genes that, in turn, induce changes in the biochemical plant machinery. Studies have shown that ROS probably require additional molecules to transduce and amplify defence signals. ROS production and anti-oxidant processes, all act in a synergistic, additive or antagonistic way, related to the control of oxidative stress. Responses to stress are not linear pathways, but are complex integrated circuits involving multiple pathways and in specific cellular compartments, tissues, and the interaction of additional cofactors and/or signalling molecules to coordinate a specified response to a given stimulus (Dombrowski, 2009). Onset of a stress triggers some (mostly unknown) initial sensors, which then activate cytoplasmic Ca2+ and protein signalling pathways, leading to stress-responsive gene expression and physiological changes (Bressan et al., 1998;

An apolar Pistacia lentiscus L. leaf extract: GC-MS metabolic profiling and evaluation of cytotoxicity and apoptosis inducing effects on SH-SY5Y and SK-N-BE(2)C cell lines.

In the course of a cytotoxicity screening of Mediterranean plants vs. neuroblastoma cells, Pistacia lentiscus was of interest. Pl-C extract, prepared from dried leaves by ultrasound assisted maceration (UAM) in chloroform, was profiled through using GC-MS techniques. To evaluate Pl-C cytotoxicity towards SH-SY5Y and SK-N-BE(2)-C cell lines, MTT, SRB and LDH assays were performed. The caspase-3 activation, DNA fragmentation, as well as micronucleation, were also evaluated. The Pl-C oxidant/antioxidant ability was estimated using different methods. The extract, rich in pentacyclic triterpenes, inhibited mitochondrial redox activity and cell viability of the tested cell lines. LDH assay established that Pl-C did not affect the cell membrane integrity. Indeed, it was able to activate caspase-3 and to cause a ladder pattern of DNA. Western blotting analysis showed that Pl-C processed caspase-3 providing two cleavage products of approximately 20 and 17-kDa, whose densitometric evaluation highlighted that Pl-C was more effective than vinblastine by 3-fold. The pro-apoptotic effect could be related to a disturbance in cell redox balance. In fact, it increased intracellular ROS production, GSSG/GSH ratio and the formation of lipoperoxidation products. The data obtained prompted to further investigate and assess the inxa0vivo efficacy of Pl-C to prevent and/or treat neuroblastoma.

DGGE analysis of buffalo manure eubacteria for hydrogen production: effect of pH, temperature and pretreatments.

Buffalo dung is a low-cost substrate with plenty of carbohydrates, an optimal carbon/nitrogen ratio, and a rich microbial flora, and could become a valuable source of biogas. Therefore, in the present study we compared the type and amount of specific eubacteria to the different configurations of pH, temperature and thermal pretreatment after fermentation in batch reactors in order to understand the suitability of buffalo manure for hydrogen production. The phylogenetic structure of the microbial community in fermentation samples was studied using denaturing gradient gel electrophoresis to generate fingerprints of 16S rRNA genes. The sequences analysis revealed abundance of the phyla Bacteroidetes and Firmicutes, and in particular of the order Clostridiales. Very active hydrogen producing bacteria belonging to Clostridium cellulosi species were identified demonstrating the suitability of this substrate to produce hydrogen. Moreover, a large fraction of 16S-rDNA amplicons could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms involved in anaerobic fermentation in digesters or bioreactors are still unclassified or unknown.

Bacterial and Archaeal Communities Influence on MethaneProduction

Bacterial and Archaeal Communities Influence on Methane Production Petronia Carillo, Claudia Carotenuto, Filomena Di Cristofaro, Carmine Lubritto, Mario Minale, Antonio Mirto, Biagio Morrone*, Stefania Papa, Pasqualina Woodrow a Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università degli Studi di Napoli,Via Vivaldi 43, 81100 Caserta, Italy b Dipartimento di Ingegneria Industriale e dell’Informazione, Seconda Università degli Studi di Napoli, Via Roma 29, 81031 Aversa (Caserta), Italy biagio.morrone@unina2.it

Metabolomics for Crop Improvement Against Salinity Stress

In the post-genomic era, increasing efforts have been done to describe the relationship between genome and phenotype in plants. It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype. Metabolites are the main readouts of gene vs environment interactions and represent the sum of all the levels of regulation in between gene and enzyme. Therefore, metabolome can be considered as the final recipient of biological information flow. Some metabolites have a very short lifetime and are indicators of specific metabolic reaction and of plant status. Indeed, it is well known that many of them are transformed during specific stresses and involved in plant stress response and resistance.

Transcription Factors and Environmental Stresses in Plants

Plants are exposed to environmental changes, which are perceived as stresses when they are quick and extreme. Drought, salt, and extreme temperatures, in particular, limit agricultural crop productivity, affecting all stages of plant growth and reproduction, and therefore strongly decreasing crop yield. Worldwide estimates show that most yield loss (70%) can be directly due to abiotic stresses. Moreover, the increasing phenomenon of enthronization and the incorrect use of agricultural land have strongly contributed to land degradation. A large number of abiotic stress-responsive genes have been reported in a variety of plants including Arabidopsis and major crops such as barley, maize, rice, and wheat. Transcriptional control of the expression of these genes is a crucial part of plant response to abiotic stresses. Therefore, recently the transcriptional mechanisms involved in the response to several abiotic stresses have been the subject of intense research, which have been productive in identifying transcription factors (TFs) as important “key or master regulators” of gene expression under stress. An increasing number of TFs have been recently described and essential transcription factor binding regions have been identified for many genes. In fact, these systems of regulation work, thanks to specific cis-elements located in the promoter regions of target genes, which are called regulons. The main regulons that respond to abiotic stresses are DREB1-CBF (dehydration-responsive element binding protein 1/C-repeat binding factor), which is involved in the cold stress response, and DREB2, which acts in ABA-independent gene expression for response to heat and osmotic stress, whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) regulon operates in gene expression depending on ABA under osmotic stress. Other regulons, such as MYB/MYC and NAC, induce or repress the expression of genes involved in abiotic stress response. In the last few years, several studies have shown that TFs are powerful tools to engineer enhanced stress tolerance in plants. Therefore, in this chapter, we will summarize the major TFs involved in crop plants’ abiotic stress signaling and responses, and the relative plants’ adaptive mechanisms at the molecular level. A major finding on molecular mechanisms that occur in stress conditions is the one-way pass for the improvement of stress tolerance in crop plants.